The affinity and rate constants for association of the specific substrate aldehyde analogue N-benzoyl-l-phenylalaninal (BzPheal) to native α-chymotrypsin (Cht) and N-methylhistidinyl-57-α-chymotrypsin (MCht) were obtained. We find an observed binding dissociation constant (k1) for BzPheal to be 2.6 X 10~5 M (in 3% Me2SO, pH 7.8) to native Cht and 3.5 X 10-6 M (in 3% Me2SO, pH 7.8) to MCht. Evidence is discussed that supports a mechanism in which the unhydrated form of BzPheal is the form that predominantly associates. Calculation of a corrected based on the solution concentrations of unhydrated BzPheal gives a K1 of 2.4 X 10-6 and 3.2 X 1(T-7 M, respectively, which is 1.35 X 103- to 1.4 X 10-4-fold smaller than substrate analogues not containing the aldehyde function. Accordingly, the high affinity of BzPheal to Cht indicates a possible transition-state-like mode of association. However, we find the pH dependence of K1 for BzPheal association to Cht shows only a fourfold increase between pH 8.0 and pH 3.0. The lack of a significant pH dependence in K1 supports the existence of a neutral hemiacetal adduct of BzPheal in Cht, rather than a more transition state like oxyanion hemiacetal adduct. The pH dependency of the second-order rate constants for BzPheal association to native Cht correlates with general base catalysis by two groups in the enzyme of pka = 4.4 and pka = 7.0. Limiting rate constants (k2/ks(lim)) for base catalysis by the groups of pka = 4.4 and 7.0 are calculated at 22 X 105 and 19 X 106 M-1 srespectively. The pH dependency of the second-order rate constant for BzPheal association to MCht correlates with general base catalysis by a single group of pka = 6.8 with k2/Ki(lim) = 21 X 105 M~s-1. Base catalysis may occur in MCht by utilization of the Nδ1 of the His-57, when the Nε2 of His-57 is methylated. The identity of the rate-limiting constants for catalysis by the monobasic form of Cht to that for BzPheal association to MCht may support the group of pka = 4.4 being the His-57 imidazole and the group of pka = 7.0 being the γcooh Asp-102. This mechanism indicates that the Asp-102 in the catalytic charge relay system only contributes a factor of 10 toward the rate of BzPheal hemiacetal formation in native Cht. However, an alternative mechanism in which the (.Nε2-methyl) nitrogen of His-57 in MCht or the protonated (Nε2) nitrogen of His-57 in native Cht acts as a general base catalyst with a Brønsted coefficient (β) of 0.07 may be possible. © 1979, American Chemical Society. All rights reserved.
